Vertical tumor thickness has great influence in the prognosis and staging of melanoma. The aim of this study was determination of the differences between melanoma tumor thickness in conventional hematoxylin and eosin (H&E) and immunohistochemical techniques. Thirty-six biopsy specimens were included in our study. For each sample, four adjacent tissue sections were stained with H&E, in addition S-100, Melan- A and HMB-45 staining was performed on the next serial sections. The mean thickness of tumor invasion was 2.16, 2.38, 2.22 and 2.29mm in H&E, S-100, HMB45 and Melan-A sections evaluation, respectively. The mean difference of the Breslow thickness between H&E and S-100 and also, between H&E and Melan-A stained slides were statistically significant (p˂0.05) while no difference was found in the tumor thickness of the H&E and HMB45 staining evaluation (p = 0.278). Greater tumor thickness was observed in 25 lesions (69.4%) with S-100, 20 lesions (55.5%) with Melan-A and 17 (47.2%) lesions in HMB-45 rather than H&E staining. Conclusively, it appears that H&E staining cannot prove the actual size of melanoma invasion in some cases and immunohistochemical examination can be a complementary method in this situations. Of the melanoma associated immunomarkers, the combination of S-100 and Melan-A staining may suffice to measure depth of tumor invasion.